Novel influenza antigens

ABSTRACT

The present invention relates to novel influenza antigens, novel immunogenic or vaccine compositions, as well as to uses of and to methods for producing said antigens and compositions. In particular, the invention relates to recombinant forms of hemagglutinin (HA) and their use in vaccine compositions for the prevention of influenza virus infections.

TECHNICAL FIELD

The present invention relates to novel influenza antigens, novelimmunogenic or vaccine compositions, as well as to uses of and tomethods for producing said antigens and compositions. In particular, theinvention relates to recombinant forms of hemagglutinin (HA) and theiruse in vaccine compositions for the prevention of influenza virusinfections.

BACKGROUND OF THE INVENTION

Influenza viruses are one of the most ubiquitous viruses present in theworld, affecting both humans and livestock. Influenza results in aneconomic burden, morbidity and even mortality, which are significant.There are three types of influenza viruses: A, B and C. Influenza viruscomprises two predominant surface antigens, the glycoproteinshemagglutinin (HA) and neuraminidase (NA), which appear as spikes at thesurface of the particles. It is these surface proteins, particularly HA,that determine the antigenic specificity of the influenza subtypes.

HA is a trimeric protein comprised of an ectodomain of identicalsubunits each of which contains two polypeptides, HA1 and HA2, linked bya disulphide bond. Each monomer is initially expressed as HA0, and issubsequently cleaved by host proteases into HA1 and HA2 subunits whichare linked via a disulfide bond. HA can be functionally divided into twodomains, the globular head and the stalk. The globular head is composedof part of HA1 and the stalk structure is composed of portions of HA1and all of HA2 (Hai et al, J. Virol, 2012 86(10): 5774-5781).

Vaccination plays a critical role in controlling influenza epidemics andpandemics. Many influenza vaccines are made by methods that involvereassortment, adaptation and growth of viruses in chicken eggs. Howeverthere are limitations with these existing methods. Not all influenzavirus strains grow well in eggs and must be adapted or viralreassortants constructed. The changes in HA during manufacture can leadto strains that differ from the circulating strains and that may offersuboptimal levels of protection. Another drawback is that those with eggallergies may show hypersensitivity to residual egg proteins in eggbased vaccines. Furthermore, egg based methods rely on an uninterruptedsupply of eggs, which can be susceptible to disruptions in supply suchas disease in poultry. There is a need for production of vaccines usingmethods that do not rely on egg supply and where vaccine proteinproduction is more stringently controlled than in egg based methods.

Recombinant forms of HA (rHA) produced in culture cells have beenproposed as an alternative source of antigen for influenza vaccines tothat sourced from eggs. However, problems maintaining immunogenicity anda regular quaternary structure of rHA have been encountered using thesemethods. There is thus still a need for alternative methods of antigensupply for influenza vaccines, that address the existing challenges.

SUMMARY OF THE INVENTION

It was found with previous efforts at producing rHA that largeaggregates of the recombinant protein formed that are not acceptable forvaccine production purposes. Additionally, the correct rosettestructure, a multimeric form of the basic trimer structure of HA, didnot always correctly form. The inventors have made a recombinanthemagglutinin (rHA) antigen which incorporates a heterologoustrimerisation domain such as a foldon, as well as a hydrophobic signalsuch as the transmembrane domain of HA and the extracellular domain(ECD) or an immunogenic portion thereof. The rHA produced by theinventors is capable of forming the correct rosette structure withoutlarge aggregates and maintains immunogenicity. These functionalproperties render the rHA potentially useful for the treatment and/orprevention of influenza infection and/or disease.

Accordingly, in a first aspect of the invention, there is provided arecombinant influenza virus hemagglutinin (HA) antigen comprising theextracellular domain of HA or an immunogenic portion thereof, ahydrophobic signal and a heterologous trimerisation domain. In a furtheraspect there is provided a HA antigen as described above wherein thehydrophobic signal is a HA transmembrane domain or an artificialhydrophobic signal.

In a further aspect there is provided a polynucleotide encoding arecombinant hemagglutinin antigen as described above.

In a further aspect there is provided an immunogenic compositioncomprising a recombinant antigen as defined above and apharmaceutically-acceptable carrier.

In a further aspect there is provided the immunogenic compositiondescribed above for use in medicine

In still a further aspect there is provided the immunogenic compositiondescribed above for use in the prevention and/or vaccination againstinfluenza disease

In still a further aspect there is provided the immunogenic compositiondescribed above for use in the prevention and/or vaccination againstinfluenza caused by a different Glade than the Glade to which theextracellular domain of the HA antigen described above belongs

In still a further aspect there is provided a method for producing arecombinant antigen as defined above comprising expressing apolynucleotide described above in a eukaryotic cell, such as a mammaliancell, e.g. a CHO cell, or an insect cell, optionally further comprisingpurifying/isolating the rHA from the eukaryotic cell

In yet a further aspect there is provided a method for prevention and/orvaccination against influenza disease, comprising the administration ofan antigen or immunogenic composition as described above to a person inneed thereof, such as a person identified as being at risk of beinginfected with influenza disease

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: The design of ECD-Foldon and ECD-TMD-Foldon used in the Examplesis shown. A. ECD-Foldon is the starting molecule, composed of the gp67secretion signal, the ectodomain of HA (ECD), a thrombin cleavage site(TCS), the trimerization domain Foldon and a histidine tail (tofacilitate purification). B. ECD-TMD-Foldon has been designed byinserting the transmembrane domain of HA (TM) between the ectodomain andthe Foldon domain.

FIG. 2: The amino acid sequences of ECD-Foldon and ECD-TMD-Foldon usedin the Examples are shown. A. ECD-Foldon is the starting molecule, andECD-TMD-Foldon has been designed by inserting the transmembrane domainof HA (TM) between the HA extracellular domain and the Foldon domain ofECD-Foldon. For information, the amino acid sequence of the full-lengthHA molecule of the same strain that has been used as comparator in thisstudy is also shown.

FIG. 3A, 3B and 3C: Electron micrographs showing the appearance ofECD-Foldon, ECD-TMD-Foldon and recombinant full-length HA, respectivelyin solution.

FIG. 4: Mice were immunized on Days 0 and 21 with ECD-Foldon (6 or 24μg), ECD-TMD-Foldon (1.5, 6 or 24 μg), a recombinant full-length HAantigen (1.5, 6 or 24 μg), split A/Indonesia/05/2005 (1.5 μg), or PBS.Blood was taken on Days 21 and 42 (3 weeks post immunizations) andlevels of anti-A/Indonesia/05/2005 antibodies were measured in mousesera by ELISA. Number in brackets indicate the number of mice in thegroup.

FIG. 5: Mice were immunized on Days 0 and 21 with ECD-Foldon (6 or 24μg), ECD-TMD-Foldon (1.5, 6 or 24 μg), a recombinant full-length HAantigen (1.5, 6 or 24 μg), split A/Indonesia/05/2005 (1.5 μg), or PBS.Blood was taken on Days 21 and 42 (3 weeks post immunizations) andlevels of anti-A/Indonesia/05/2005 antibodies were measured in mousesera by hemagglutination inhibition assay (HI). Numbers in bracketsindicate the number of mice in the group.

FIG. 6: Mice were immunized on Days 0 and 21 with ECD-Foldon (6 or 24μg), ECD-TMD-Foldon (1.5, 6 or 24 μg), a recombinant full-length HAantigen (1.5, 6 or 24 μg), split A/Indonesia/05/2005 (1.5 μg), or PBS.Blood was taken on Days 21 and 42 (3 weeks post immunizations) andlevels of anti-A/Indonesia/05/2005 neutralizing antibodies were measuredin mouse sera by neutralization assay. Numbers in brackets indicate thenumber of mice in the group.

FIG. 7: Mice were immunized on Days 0 and 21 with ECD-Foldon (6 or 24μg), ECD-TMD-Foldon (1.5, 6 or 24 μg), a recombinant full-length HAantigen (1.5, 6 or 24 μg), split A/Indonesia/05/2005 (1.5 μg), or PBS.Blood was taken on Days 21 and 42 (3 weeks post immunizations) andlevels of anti-A/Indonesia/05/2005 antibodies were measured in mousesera by ELISA and hemagglutination assay. Graph shows the ratios betweenELISA and HI values. Numbers in brackets indicate the number of mice inthe group. Asterisk (*) indicates that ratio could not be calculated asat least one of the two values was below the cut-off of the assay.

FIG. 8: Mice were immunized on Days 0 and 21 with ECD-Foldon (6 or 24μg), ECD-TMD-Foldon (1.5, 6 or 24 μg), a recombinant full-length HAantigen (1.5, 6 or 24 μg), split A/Indonesia/05/2005 (1.5 μg), or PBS.Blood was taken on Days 21 and 42 (3 weeks post immunizations) andlevels of anti-A/Indonesia/05/2005 antibodies were measured in mousesera by ELISA and levels of neutraliziong antibodies by neutralizationassay. Graph shows the ratios between ELISA and neutralizing values.Number in brackets indicate the number of mice in the group. Asterisk(*) indicates that ratio could not be calculated as at least one of thetwo values was below the cut-off of the assay.

DETAILED DESCRIPTION

Provided herein is a recombinant influenza virus hemagglutinin (rHA)antigen comprising or consisting of the extracellular domain of HA(ectodomain, ECD) or an immunogenic portion thereof, a hydrophobicsignal such as a HA transmembrane domain (TMD) and a heterologoustrimerisation domain.Recombinant Influenza Virus HA (rHA)A rHA comprises or is encoded by one or more nucleic acids that arederived from a nucleic acid which was artificially constructed. Forexample, the nucleic acid can comprise, or be encoded by, a clonednucleic acid formed by joining heterologous nucleic acids.The rHA includes hemagglutinin-derived sequences (such as the ECD andTMD) and may include other non-hemagglutinin derived sequences, forexample a non-hemagglutinin derived heterologous trimerisation domain.Typically, the hemagglutinin-derived sequences are in the order thatthey appear in naturally derived hemagglutinin and the trimerisationdomain occurs towards, or at the C-terminal, for example in theC-terminal half of the rHA/C-terminal to the ECD. The rHA of theinvention, may in particular consist or comprise of the ECD of HA or animmunogenic portion thereof followed by a HA TMD, followed by aheterologous trimerisation domain, in that order, wherein thetrimerisation domain is in the C-terminal half of the rHA/C-terminal tothe ECD.The rHA antigen of the invention may be fused to or contain furtherpolypeptide other than ECD, TMD and trimerisation domain. The sequenceencoding the further polypeptide optionally includes additional featuressuch as a flexible linker between the HA derived sequences and otherheterologous amino acid sequences. The linkers can facilitate theindependent folding of the HA domains and other heterologous sequences.The linker may be an amino acid sequence that is synthesized as part ofa recombinant fusion protein. In other embodiments, a chemical linker isused to connect synthetically or recombinantly produced subsequences.Such flexible linkers are known to those skilled in the art.In addition to flexible linkers, or alternatively, the fusion proteinsoptionally include polypeptide subsequences from proteins which areunrelated to hemagglutinin, e.g. a sequence with affinity to a knownantibody to facilitate affinity purification and/or detection. Suchdetection and purification-facilitating domains include, but are notlimited to, metal chelating peptides such as polyhistidine tracts andhistidine-tryptophan modules that allow purification on immobilizedmetals and protein A domains that allow purification on immobilizedimmunoglobulin. Examples include heterologous fusion sequences encodinggD tags, c-Myc epitopes, poly-histidine tags, Fluorescent proteins (e.g.GFP), beta-galactosidase protein or glutathione S transferase or anyother sequence useful for detection or purification of the fusionprotein expressed in or on a cell. Preferably, the further polypeptidesequence is a polyhistidine tag, such as a six histidine tag. Theinclusion of a cleavable linker sequence between the purification domain(e.g. polyhistidine tag) and rHA antigen may be useful to facilitatepurification. For example an enzyme cleavage site, such as a thrombincleavage site may be included between the further polypeptide and therest of the rHA sequences. A cleavable linker sequence, for example anenzyme cleavage site such as a thrombin cleavage site may alternativelyor additionally be included between the trimerisation domain and therest of the rHA sequences. This can allow the trimerisation domain to beremoved in the final rHA. Hence, the rHA antigen of the invention mayconsist of or comprise (in order) the ECD of HA or an immunogenicportion thereof, a HA TMD, a heterologous trimerisation domain, apurification tag (e.g. polyhistidine tag) and optionally a cleavablelinker sequence i) between the purification tag and the rest of the rHAand/or ii) between the trimerisation domain and the HA TMD.The gene or construct encoding the rHA antigen may include a signalpeptide. Typically, the signal peptide is appropriate for the host cellin which the rHA is expressed. In one embodiment, the natural signalpeptide sequence in hemagglutinin is deleted and replaced with abaculovirus signal peptide, for example secretion signal gp67 (Whitfordet al 1989, J. Virol. 63, 1393-1399), for proper expression in insectcells. The gene containing rHA antigen and baculovirus signal peptidecan be introduced into a baculovirus expression vector so that thebaculovirus promoter directs the transcription of the fusion proteins ininfected insect cells. The signal peptide directs the translation of therHA antigen into the insect cell glycosylation pathway and is notpresent on the mature protein.Accordingly, nucleic acid sequence encoding the rHA antigen of theinvention may consist of or comprise sequence encoding the ECD of HA oran immunogenic portion thereof, a HA TMD, a heterologous trimerisationdomain (e.g. foldon), a purification tag (e.g. polyhistidine tag) and asignal peptide (e.g. a baculovirus signal peptide), such as in theorder: a signal peptide (e.g. a baculovirus signal peptide), the ECD ofHA or an immunogenic portion thereof, a HA TMD, a heterologoustrimerisation domain (e.g. foldon), a purification tag (e.g.polyhistidine tag). As another example, the nucleic acid encoding therHA antigen of the invention may consist of or comprise sequenceencoding (in order): a signal peptide (e.g. a baculovirus signalpeptide), the ECD of HA or an immunogenic portion thereof, a HA TMD, acleavable linker sequence (e.g. TCS), a heterologous trimerisationdomain (e.g. foldon), a purification tag (e.g. polyhistidine tag).The HA sequences (e.g. the ECD (or immunogenic portion thereof) and TMD)of the rHA antigen may be from any type or subtype (e.g. H1 to H16) ofinfluenza strain. In one embodiment, the HA sequence of the HA antigenis from a strain selected from the group consisting of: an H1, an H2, anH3, an H5, an H7 and an H9 subtype strain. Preferably, the HA antigen isfrom an H5 strain. The sequences for the ECD (or immunogenic portionthereof) and TMD can be from the same source/strain/type/subtype ofinfluenza.In some embodiments, the HA has a sequence that is identical to HA froma pandemic strain. By pandemic strain, it is meant a new influenza virusagainst which the large majority of the human population has noimmunity. Typically, the WHO identifies and publicises such pandemicstrains. Suitable pandemic strains are, for example H5N1, H9N2, H7N7,H7N9, H2N2, H7N1, H7N3, H10N7, H5N2 and H1N1. Alternatively, the HAsequence is identical or derived from naturally occurring HA from anon-pandemic strain. For example, the non-pandemic strains may bestrains identified by WHO as circulating seasonal influenza virusstrains or strains identified by WHO as having the potential for causingan epidemic for the subsequent influenza season. Such strains may forexample be 1) H1N1, H3N2 influenza A type strains and 2) one or two Btype influenza strains (e.g. from Victoria and/or Yamagata lineages).For example, the rHA antigen of the invention may comprise i) an aminoacid sequence comprising the ECD of HA (e.g. SEQ ID NO: 7) or animmunogenic portion or derivative thereof, ii) amino acid sequencecomprising an HA TMD (e.g. SEQ ID NO: 5) and iii) the heterologoustrimerisation domain (foldon) shown in SEQ ID NO: 9 or a derivative ofthis sequence that maintains the ability to induce rHA monomers to formtrimers. In particular, the ECD of HA may be derived from an H5 virus(e.g. an H5N1 virus), such as that shown in SEQ ID NO: 7. Both the ECDand/or the TMD of HA may be derived from H5 virus such as an H5N1 virus.For example, the rHA antigen may comprise i) SEQ ID NO: 7 or animmunogenic portion or derivative thereof and/or ii) SEQ ID NO: 5 orderivative thereof that retains the ability to orientate the HA trimersinto rosette structures and maintains the immunogenicity of HA. Inparticular, the rHA antigen may comprise or consist of the amino acidsequence shown in SEQ ID NO: 1.The nucleic acid sequence encoding the rHA antigen may comprise i)nucleic acid encoding the ECD of HA (e.g. SEQ ID NO: 8), or a fragmentor derivative of this sequence encoding an immunogenic portion of the HAECD, ii) nucleic acid sequence (e.g. SEQ ID NO: 6) encoding the TMD ofHA or a fragment or derivative of this sequence that encodes a TMD thatretains the ability to orientate the HA trimers into rosette structuresand maintains the immunogenicity of HA and iii) SEQ ID NO: 10 encodingfoldon or a derivative of this sequence that maintains the ability toinduce expressed rHA monomers to form trimers.The ECD and/or TMD sequence may be derived from an H5 virus e.g an H5N1virus. For example, in one embodiment the nucleic acid sequence encodingthe rHA antigen comprises i) SEQ ID NO: 8 or a fragment or derivative ofthis sequence encoding an immunogenic portion of the HA ECD and ii) SEQID NO: 6, or a fragment or derivative of this sequence that encodes aTMD that retains the ability to orientate the HA trimers into rosettestructures and maintains the immunogenicity of HA. In particular,nucleic acid encoding the rHA antigen of the invention may consist orcomprise of the nucleic acid sequence shown in SEQ ID NO: 2.In another instance, nucleic acid encoding the rHA antigen of theinvention may comprise any nucleic acid sequence encoding an HA ECD, anysequence encoding a HA TMD and the foldon sequence shown in SEQ ID NO:10 or a derivative of this sequence that maintains the ability to induceexpressed rHA monomers to form trimers. In a further instance, nucleicacid encoding the rHA antigen of the invention may comprise i) anynucleic acid sequence encoding an HA ECD, ii) any nucleic acid sequenceencoding a heterologous trimerisation domain and iii) SEQ ID NO: 6encoding the TMD of HA or a fragment or derivative of this sequence thatencodes a TMD that retains the ability to orientate the HA trimers intorosette structures and maintains the immunogenicity of HA. In a yetfurther instance, nucleic acid encoding the rHA of the invention maycomprise i) SEQ ID NO: 8 encoding the ECD of HA, or a fragment orderivative of this sequence encoding an immunogenic portion of the HAECD, ii) any nucleic acid sequence encoding a heterologous trimerisationdomain and iii) any nucleic acid encoding a TMD of HA. In a yet furtherinstance still, nucleic acid encoding the rHA antigen of the inventionmay comprise i) any sequence encoding an ECD of HA, ii) the foldonsequence shown in SEQ ID NO: 10 or a derivative of this sequence thatmaintains the ability to induce expressed rHA monomers to form trimersand iii) any nucleic acid encoding a TMD of HA.Preferably, the recombinant influenza virus hemagglutinin (HA) antigenof the invention lacks the intracellular domain of influenzahemagglutinin, e.g. the intracellular domain represented by SEQ ID NO:3. Also provided is a nucleic acid sequence encoding the recombinantinfluenza virus hemagglutinin (HA) antigen of the invention that lackssequence encoding the intracellular domain of influenza hemagglutinin,e.g. the intracellular domain represented by SEQ ID NO:4.

Extracellular Domain (ECD) or Immunogenic Portion Thereof

The extracellular domain (or ectodomain, ECD) component of HA is presentin wild-type HA protein at the cell surface. The rHA of the inventionmay comprise a full length ECD, or an immunogenic portion thereof. TheECD or immunogenic portion thereof, may in some embodiments be a variantor derivative of wild-type HA protein (for example containing amino acidsubstitutions, deletions or additions).The immunogenic portion thereof, may include one or more regions of HAfor which it is desired to direct an immune response. Such regions mayinclude known conserved and/or variable epitopes of hemagglutinin thatelicit neutralising antibodies upon vaccination. Preferably, the portionthereof is capable of proper folding to interact in a hemagluttinationassay.For instance, the ECD may consist or comprise the HA1 and/or HA2 regionof HA. Alternatively, the ECD may consist or comprise the head and/orstalk region of HA. An ECD may consist or comprise the HA2 subunit and aportion of the HA1 subunit, that together form the stalk region of HA.The ECD may consist of the stalk region of HA, a so-called “headless”form of HA. In particular, in some embodiments, the HA antigen may lackthe HA head, or part of the head, such as more than 25% e.g. more than50%, such as more than 75% of the amino acid residues of the head, orlack the HA1 part of the head. Alternatively, the HA sequence may lackthe HA stalk, or part of the stalk, such as more than 25%, e.g. morethan 50%, such as more than 75% of the amino acid residues of the stalk,or lack the HA2 part of the stalk.The terms “HA1” refers to the region of the HA protein including aminoacid residues from approximately 1-330 of the extracellular domain of HAprotein. HA1 comprises all residues that are N-terminal to the HA1/HA2cleavage peptide of the precursor HA0 protein, including the receptorbinding domain of the HA protein.The term “HA2” refers to the region of the HA protein including aminoacid residues from approximately 331-504 of the HA0 haemagglutinpolypeptide. Of note, these residues within the HA2 chain are commonlynumbered independently of those in HAL such that HA2 residues may benumbered consecutively 1-174. The HA2 chain comprises all residues thatare C-terminal to the HA1/HA2 cleavage peptide of the precursor HA0protein, including the hydrophobic peptide responsible for insertionwithin the host cell membrane during the process of membrane fusion. Theterm “HA stalk” refers to the region of the HA protein includingresidues from approximately 1-42 and 274-330 of the HA1 chain as well asresidues (1-174) of the HA2 chain. The stalk is located in themembrane-proximal region of the HA, directly beneath the vestigialesterase domain of the HA1 globular head.The term “HA head” refers to a globular head region of the HA proteinexcluding the transmembrane domain and any intracellular region, whichis composed of part of HA1 and that contains a sialic acid bindingpocket that mediates virus attachment to the host cell. See for exampleHai et al (J. Virol, 2012 86(10): 5774-5781).Numbering of the amino acid sequence of the ECD is consecutive from theamino (N-) terminal to the carboxyl (C-) terminal residue, such thatposition 1 corresponds to the residue at the N-terminus of eachsub-domain in the wild type HA as found in virions. As such, anyadditional engineered residues at the N-terminus, such as theheterologous sequences described herein, and those introduced as part ofthe expression strategy or for the purposes of solubilisation orpurification, are numbered in the reverse order (i.e. from —C) toN-terminal) from position 1, starting with position 0 (e.g. 0, -1, -2,etc).The ECD sequence may be derived from any type or subtype (e.g. H1 toH16) of influenza strain. In one embodiment, the HA ECD sequence is froma strain selected from the group consisting of: an H1, an H2, an H3, anH5, an H7 and an H9 subtype strain. Preferably, the ECD is from an H5strain such as an H5N1 strain.For example, the HA ECD may comprise or consist of the amino acidsequence shown in SEQ ID NO: 7 or a fragment or derivative of thissequence that contains an immunogenic portion of HA. Nucleic acidencoding the HA ECD may comprise or consist of the nucleic acid sequenceshown in SEQ ID NO: 8 or a fragment or derivative of this sequenceencoding an immunogenic portion of the HA ECD.The term “hydrophobic signal” refers to a stretch of at least 5 or atleast 6 hydrophobic aminoacids, or refers to an overall structure wherethe hydrophobic aminoacids are surface exposed. Hydrophobic signals maybe natural or artificial. Natural hydrophobic signals are present incellular or viral transmembrane proteins. Any natural or artificialhydrophobic signals must retain the original function in the protein,e.g. in case of HA proteins, the hydrophobic signal must have theability to orientate the HA trimers into rosette structures. Thehydrophobic signal naturally present in HA proteins is named thetransmembrane domain.

Transmembrane Domain (TMD)

The transmembrane domain may consist or comprise a full length TMD froma wild-type HA protein or a truncation or derivative thereof. Anytruncation or derivative thereof must retain the ability to orientatethe HA trimers into rosette structures and maintain the immunogenicityof HA. The proper rosette structure can be detected using techniqueswell known to those skilled in the art such as electron microscopy.The transmembrane domain may also be derived from any type or type orsubtype (e.g. H1 to H16) of influenza strain. In one embodiment, the HATMD sequence is from a strain selected from the group consisting of: anH1, an H2, an H3, an H5, an H7 and an H9 strain. Preferably, the TMD isfrom an H5 strain such as an H5N1 strain. The TMD may be derived fromthe same or a different strain to the ECD sequences. The TMD may beeither homologous or heterologous to the ECD. For example, the HA TMDmay comprise or consist of the amino acid sequence shown in SEQ ID NO: 5or a fragment or derivative of this sequence that retains the ability toorientate HA trimers into rosette structures and maintains theimmunogenicity of HA. Nucleic acid encoding the HA TMD may comprise orconsist of the nucleic acid sequence shown in SEQ ID NO: 6 or a fragmentor derivative of this sequence encoding a TMD that retains the abilityto orientate HA trimers into rosette structures and maintains theimmunogenicity of HA.

Trirnerisation Domain

A suitable trimerisation domain is one that induces rHA monomers to formtrimers. Preferably the trimerisation domain is or is derived from thenatural trimerisation domain of T4 phage fibritin “foldon”. A 29 aminoacid foldon sequence may be used which forms a β-propeller structurecomprising the C terminus of the fibritin domain of the T4bacteriophage. Other suitable trimerisation domains includechloramphenicol acetyl transferase (CAT) and a leucine zippertrimerisation motif derived from the yeast transcription activator GCN4.Most preferably, the trimerisation domain, such as foldon, is placed atthe C terminus of the HA extracellular domain and TMD (e.g. stemdomain). Typically, the trimerisation domain is fused via a short linkerregion to the HA sequence. The region between the trimerisation domainand HA sequence may include a cleavable linker sequence, so it ispossible to isolate the HA sequence from the trimerisation at laterstages. Thus, the HA sequence (which includes the ECD and TMD) may belinked (e.g. in order), optionally via a linker sequence, toheterologous sequence comprising a protease cleaveage site, thetrimerisation domain and a purification tag such as histidine tag to aidin purification. Such heterologous trimerisation domains may be linkedto HA sequences by techniques known in the art, such as molecularcloning.For example, the trimerisation domain may comprise or consist of thefoldon amino acid sequence shown in SEQ ID NO: 9 or a derivative of thissequence that maintains the ability to induce rHA monomers to formtrimers. Nucleic acid encoding the trimerisation domain may consist ofor comprise SEQ ID NO: 10 or a derivative of this sequence thatmaintains the ability to induce expressed rHA monomers to form trimers,e.g. as evaluated by electron microscopy.

Methods of Preparing rHA

Use of recombinant DNA technology to produce influenza vaccines offersseveral advantages. This includes avoiding the steps of adaptation andpassage of infectious viruses in eggs and production of more highlypurified protein under safer and more stringently controlled conditions.Moreover, no virus inactivation step has to be included. Any suitablecloning and expression system may be used to recombinantly produce therHA antigen.Nucleotide sequences encoding the rHA antigens of the invention may besynthesized, and/or cloned and expressed according to techniques wellknown to those in the art. See for example, Sambrook, et al, MolecularCloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989). In some embodiments, the polynucleotidesequences will be codon optimised for a particular recipient host cellusing standard methodologies. For example, a DNA construct encoding ahemagglutinin sequence can be codon optimised for expression in otherhosts, e.g. bacteria, mammalian or insect cells. Suitable host cells mayinclude bacterial cells such as E. Coli, fungal cells such as yeast,insect cells such as Drosophila S2, Spodoptera Sf9, Sf00+ or Hi-5 andanimal cells such as CHO.Hemagglutinin sequences may be produced by standard recombinant methodsknown in the art, such as polymerase chain reaction (PCR) or reversetranscriptase PCR, reverse engineering or the DNA can be synthesized.For PCR, primers can be prepared using hemagglutinin nucleotidesequences that are available in publicly available databases.Polynucleotide constructs may be assembled from PCR cassettes andsequentially cloned into a vector containing a selectable marker forpropagation in a host cell.A recombinant vector can then be introduced into the host cell byinjection, transfection or electroporation or other methods (forexample, calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation).Commercial transfection reagents such as Lipofectamine (Invitrogen,Carlsbad, Calif.) are also available.The rHA antigen can be recovered and purified from recombinant cellcultures by methods known in the art, including anion and/or cationexchange chromatography, affinity chromatography. Techniques such asSDS-PAGE can be used to analyse fractions of protein eluted from theseseparation/purification techniques. Such methods are well known to thoseskilled in the art and will not be presented in detail here.Proper folding of the rHA antigen can be determined for example by usingthe red blood cell hemagglutination assay, by the ability of the proteinto bind an influenza receptor, by immunogenicity testing in a hostanimal and/or determination of the ability of the protein to assume anappropriate quaternary structure such as rosette formation.Preferably a baculovirus expression system is used, which is describedbelow.

Baculovirus Expression System

When using a baculovirus expression system, the rHA antigen of interesttogether with any heterologous sequence (for example HA sequencestogether with the trimerisation domain) can be inserted into abaculovirus expression vector. Recombinant baculovirus that expressforeign genes can be made by homologous recombination betweenbaculovirus DNA and plasmids containing the insert, using well knowntechniques. The insertion may, for example be made so that the insert isunder the transcription control of the polyhedron promoter, thebaculovirus promoter.Example of baculovirus expression vectors including a vector derivedfrom the well characterised Autographa californica Nuclear PolyhedrosisVirus (AcNPV) which replicates efficiently in susceptible culturedinsect cells.Any suitable insect host cell can be used to produce the recombinant HAantigen, including but not limited to Sf900+, Sf9 or Hi-5 cells.Preferably, the insect host cell is Sf9 or Hi-5. Cells may be propagatedwith culture medium and culture conditions known to be suitable for theselected host cell. Cells may be propagated for example in monolayer orin free suspension culture.rHA protein may then be isolated from the host cells using methods wellknown in the art. For example, the cell culture may be centrifuged, thesupernatant collected and run through appropriate anionic and/orcationic exchange columns to purify the protein. Techniques such asSDS-PAGE and/or immunoblotting may be used to check the identity andintegrity of proteins. A fraction of interest containing rHA may then befurther purified, for example by passing through a nickel column so thatrHA displaying a histidine tag binds the nickel in the column.The extent of trimerisation and/or multimerisation (e.g. rosetteformation) may be tested for example by crosslinking of HA using asuitable crosslinking agent and then use of gel migration techniques, aswell known in the art. Other techniques include electron microscopy orspectroscopy based techniques that are also well known to those skilledin the art.

Rosette Formation

In one embodiment, the rHA antigen of the present invention is found inthe form of rosettes. The rosettes consist of multimers of the HAtrimers having a rosette-like structure. The rosettes are visable forexample in an electron microscope. More quantitative techniques can alsobe used to measure rosette formation, including gel filtration andspectroscopy based techniques. Rosettes generally comprise 20-100 HAtrimers/particle. The particle size of the rosette structures range from20-40 nanometers (nm) in length. HA sediments as a rosette comprised of5-6 trimers over the pH range 7.4-7.5 (Remeta et al 2002, Biochem. 41,2044-2054). It is thought that the hydrophilic C-terminal portions of HAtrimers concentrate together into a core region from which thehydrodrophic regions splay out like a snowflake or rosette structure.

Immunogenic Composition

In a further aspect, an immunogenic composition comprising an HA antigenof the invention and a pharmaceutically acceptable carrier is provided.In one embodiment, said composition further comprises an adjuvant.Preferably, the adjuvant is an oil-in-water emulsion adjuvant. Oil inwater emulsion adjuvants, such as MF59 or AS03 are well known in the artand are described below.In one embodiment, the composition is monovalent, i.e. only comprisesone influenza HA. In alternative embodiments, the composition ismultivalent, i.e. comprises multiple influenza virus antigens. Forexample, the composition may be bivalent, trivalent or quadrivalent,e.g. may contain two or three seasonal strains with the rHA of theinvention.

Adjuvant

In one embodiment, an immunogenic composition of the invention comprisesan adjuvant. In particular, the adjuvant may be an emulsion, such as anoil-in-water emulsion. Optionally, other immunostimulants may be presentin the oil-in-water emulsion. In a specific embodiment, an oil-in-wateremulsion comprises a metabolisable oil, non-toxic oil such as squaleneor squalane, optionally a tocol such as tocopherol in particular alphatocopherol and an emulsifier (or surfactant) such as the non-ionicsurfactant polyoxyethylene sorbitan monooleate (TWEEN-80™ or polysorbate80™). Mixtures of surfactants can be used such as polyoxyethylenesorbitan monooleate/sorbitan trioleate (SPAN 85™) mixtures, orpolyoxyethylene sorbitan monooleate/t-octylphenoxypolyethoxyethanol(TRITON X100™) mixtures.Tocols (e.g. Vitamin E) are also used in oil emulsions adjuvants(EP0382271B1; U.S. Pat. No. 5,667,784; WO95/17210). Tocols used in oilemulsions (optionally oil-in-water emulsions) may be formulated asdescribed in U.S. Pat. No. 5,650,155A; U.S. Pat. No. 5,667,784A;EP0382271B1, in that the tocols may be dispersions of tocol droplets,optionally comprising an emulsifier, of optionally less than 1 micron indiameter. Alternatively, the tocols may be used in combination withanother oil, to form the oil phase of an oil emulsion. Examples of oilemulsions which may be used in combination with the tocol are describedherein, such as the metabolisable oils described above. In anoil-in-water emulsion, the oil and emulsifier should be in an aqueouscarrier. The aqueous carrier may be, for example, phosphate bufferedsaline or a citrate buffer. One example of a tocol-containingoil-in-water emulsion is AS03.A preferred oil-in-water emulsion comprises a metabolisable oil, such assqualene, Tween 80 and optionally alpha tocopherol. Additionally, theoil-in-water emulsion may contain Span 85™ and/or lecithin.In one aspect, the oil-in-water emulsion has one of the followingcompositions:

-   -   From 0.5 to 11 mg squalene, from 0.05 to 5% polyoxythylene        sorbitan monooleate (TWEEN-80™ or POLYSORBATE 80™) and        optionally, from 2 to 12% alpha-tocopherol; or    -   About 5% squalene, about 0.5% polyoxyethylene sorbitan        monooleate (TWEEN-80™ or POLYSORBATE 80™) and about 0.5%        sorbitan trioleate (SPAN 85™). This adjuvant is called MF59.        An alternative adjuvant that may be used with the compositions,        vaccine or antigen according the present invention, comprises an        immunologically active saponin fraction derived from the bark of        Quillaja Saponaria Molina (e.g. QS21) presented in the form of a        liposome and a lipopolysaccharide (e.g. 3D-MPL), optionally        further including a sterol (cholesterol). In one embodiment, the        adjuvant comprises or consists of a saponin (e.g. QS21)        presented in the form of a liposome, a lipid A derivative such        as 3D-MPL and a sterol (e.g. cholesterol). The liposomes        suitably contain a neutral lipid, for example,        phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) or        dilauryl phosphatidylcholine. The liposomes may also contain a        charged lipid which increases the stability of the liposome-QS21        structure for liposomes composed of saturated lipids. An example        of such an adjuvant is AS01, which comprises 3D-MPL and QS21 in        a quenched form with cholesterol, and can be made as described        in WO96/33739. Either the ASO1B or ASO1E forms of this adjuvant        may be used. The AS01 B adjuvant comprises liposomes, which in        turn comprise dioleoyl phosphatidylcholine (DOPC), cholesterol        and 3D-MPL (in an amount of approximately 1000 micrograms DOPC,        250 micrograms cholesterol and 50 micrograms 3D-MPL per vaccine        dose), QS21 (50 micrograms/dose), phosphate NaCl buffer and        water to a volume of 0.5 ml.        The ASO1E adjuvant comprises the same ingredients than AS01 B        but at a lower concentration in an amount of approximately 500        micrograms DOPC, 125 micrograms cholesterol, 25 micrograms        3D-MPL and 25 micrograms QS21, phosphate NaCl buffer and water        to a volume of 0.5 ml.

Vaccination Regimes, Dosing and Efficacy Criteria

Suitably, the immunogenic compositions for use according to the presentinvention are a standard 0.5 ml injectable dose in most cases, andcontain 15 μg or less, of hemagglutinin antigen component from aninfluenza virus strain, as measured by single radial immunodiffusion(SRD) (J. M. Wood et al.: J. M m Biol. Stand. 5 (1977) 237-247; J. M.Wood et al., J. Biol. Stand. 9 (1981) 317-330). Suitably the vaccinedose volume will be from 0.25 ml to 1 ml, in particular a standard 0.5ml, or 0.7 ml vaccine dose volume. Slight adaptation of the dose volumewill be made routinely depending on the HA concentration in the originalbulk sample and depending also on the delivery route with smaller dosesbeing given by the intranasal or intradermal route. Suitably saidimmunogenic compositions for use according to the invention contain alow amount of HA antigen—e.g. any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 μg of HA per influenza virus strain or which does not exceed15 μg of HA per strain. Said low amount of HA amount may be as low aspractically feasible provided that it allows to formulate a vaccinewhich meets the international e.g. EU or FDA criteria for efficacy, asdetailed below (see Table 1 and the specific parameters as set forth). Asuitable low amount of HA is from 1 to 7.5 μg of HA per influenza virusstrain, suitably from 3.5 to 5 μg, such as 3.75 or 3.8 μg of HA perinfluenza virus strain, typically about 5 μg of HA per influenza virusstrain. Another suitable amount of HA is from 0.1 to 5 μg of HA perinfluenza virus strain, suitably from 1.0 to 2 μg of HA per influenzavirus strain, such as 1.9 μg of HA per influenza virus strain.The influenza medicament (e.g. immunogenic composition) of the inventionsuitably meets certain international criteria for vaccines. Standardsare applied internationally to measure the efficacy of influenzavaccines.Serological variables are assessed according to criteria of the EuropeanAgency for the Evaluation of Medicinal Products for human use(CHMP/BWP/214/96, Committee for Proprietary Medicinal Products (CPMP).Note for harmonization of requirements for influenza vaccines, 1997.CHMP/BWP/214/96 circular N° 96-0666:1-22) for clinical trials related toannual licensing procedures of influenza vaccines (Table below).

TABLE 1 CHMP criteria 18-60 years >60 years Seroconversionrate* >40% >30% Conversion factor** >2.5 >2.0 Protectionrate*** >70% >60% *Seroconversion rate is defined as the proportion ofsubjects in each group having a protective post-vaccination titre ≥1:40.The seroconversion rate simply put is the % of subjects who have an HItitre before vaccination of <1:10 and ≥1:40 after vaccination. However,if the initial titre is ≥1:10 then there needs to be at least a fourfoldincrease in the amount of antibody after vaccination. **Conversionfactor is defined as the fold increase in serum HI geometric mean titres(GMTs) after vaccination, for each vaccine strain. ***Protection rate isdefined as the proportion of subjects who were either seronegative priorto vaccination and have a (protective) post-vaccination HI titre of≥1:40 or who were seropositive prior to vaccination and have asignificant 4-fold increase in titre post-vaccination; it is normallyaccepted as indicating protection.The requirements are different for adult populations (18-60 years) andelderly populations (>60 years). For interpandemic influenza vaccines,at least one of the assessments (seroconversion factor, seroconversionrate, seroprotection rate) should meet the European requirements, forall strains of influenza included in the vaccine. The proportion oftitres equal or greater than 1:40 is regarded most relevant becausethese titres are expected to be the best correlate of protection (Beyeret al. (1998) Clin Drug Invest 15:1).

The compositions for use according to the present invention suitablymeet at least one such criteria for the influenza virus strain includedin the composition (one criteria is enough to obtain approval), suitablyat least two, or typically at least all three criteria for protection.Suitably the above response(s) is(are) obtained after one dose, or aftertwo doses.

Methods of Treatment

In a further embodiment, the HA antigen or immunogenic compositioncomprising said antigen is for use in medicine, such as for use in theprevention of, or vaccination against, influenza e.g. administered to aperson (e.g. subject) at risk for influenza infection.In a yet further embodiment, the HA antigen or immunogenic compositioncomprising said antigen is for use in the prevention of influenza causedby a different Glade than the Glade on which the HA antigen was based.For example, a H5N1 Glade 1 HA antigen could be used for protectionagainst influenza caused by a non-Glade 1 virus e.g. a H5N1 Glade 2virus. In a further aspect, there is provided a method of preventionand/or treatment against influenza disease, comprising theadministration of an antigen or immunogenic composition as describedherein to a person in need thereof, e.g. to a person (e.g. subject) atrisk for influenza infection, e.g. an elderly person (age 50 or over,particularly age 65 or over). In one embodiment of the above describedmethod or use, less than 15 micrograms, such as from 3.75 to 10micrograms of HA is administered per dose.In one aspect, the invention provides the rHA of the invention at a doseof below 10 micrograms, or below 8 micrograms, or from 1-7.5 micrograms,or from 1-5 micrograms of rHA for use in a vaccination regimen for theprevention of influenza, wherein the hemagglutinin sequences are from,or derived from a strain of influenza identified by an internationalorganisation such as the WHO that monitors outbreaks of influenza virus,as associated with a pandemic outbreak or as having the potential to beassociated with a future pandemic outbreak.

Routes of Administration

The composition of the invention may be administered by any suitabledelivery route, such as intradermal, mucosal (e.g. intranasal), oral,intramuscular or subcutaneous. Other delivery routes are well known inthe art.The intramuscular delivery route is particularly suitable for theadjuvanted influenza composition. The composition according to theinvention may be presented in a monodose container, or alternatively, amultidose container, particularly suitable for a pandemic vaccine.In this instance an antimicrobial preservative such a thiomersal may bepresent to prevent contamination during use. A thiomersal concentrationof 5 μg/0.5 ml dose (i.e. 10 μg/ml) or 10 μg/0.5 ml dose (i.e. 20 μg/ml)is suitably present. A suitable IM delivery device could be used such asa needle-free liquid jet injection device, for example the Biojector2000 (Bioject, Portland, Oreg.). Alternatively a pen-injector device,such as is used for at-home delivery of epinephrine, could be used toallow self administration of vaccine. The use of such delivery devicesmay be particularly amenable to large scale immunization campaigns suchas would be required during a pandemic.Intradermal delivery is another suitable route. Any suitable device maybe used for intradermal delivery, for example short needle devices. Suchdevices are well known in the art. Intradermal vaccines may also beadministered by devices which limit the effective penetration length ofa needle into the skin, such as those described in WO99/34850 andEP1092444, incorporated herein by reference, and functional equivalentsthereof. Also suitable are jet injection devices which deliver liquidvaccines to the dermis via a liquid jet injector or via a needle whichpierces the stratum corneum and produces a jet which reaches the dermis.Also suitable, are ballistic powder/particle delivery devices which usecompressed gas to accelerate vaccine in powder form through the outerlayers of the skin to the dermis. Additionally, conventional syringesmay be used in the classical mantoux method of intradermaladministration.Another suitable administration route is the subcutaneous route. Anysuitable device may be used for subcutaneous delivery, for exampleclassical needle. Suitably, a needle-free jet injector service is used.Such devices are well known in the art. Suitably said device ispre-filled with the liquid vaccine formulation.Alternatively the vaccine is administered intranasally. Typically, thevaccine is administered locally to the nasopharyngeal area, suitablywithout being inhaled into the lungs. It is desirable to use anintranasal delivery device which delivers the vaccine formulation to thenasopharyngeal area, without or substantially without it entering thelungs.Suitable devices for intranasal administration of the vaccines accordingto the invention are spray devices. Suitable commercially availablenasal spray devices include Accuspray™ (Becton Dickinson). Nebulisersproduce a very fine spray which can be easily inhaled into the lungs andtherefore does not efficiently reach the nasal mucosa. Nebulisers aretherefore not preferred.Suitable spray devices for intranasal use are devices for which theperformance of the device is not dependent upon the pressure applied bythe user. These devices are known as pressure threshold devices. Liquidis released from the nozzle only when a threshold pressure is applied.These devices make it easier to achieve a spray with a regular dropletsize. Pressure threshold devices suitable for use with the presentinvention are known in the art and are described for example in WO91/13281 and EP 311 863 B and EP 516 636, incorporated herein byreference. Such devices are commercially available from Pfeiffer GmbHand are also described in Bommer, R. Pharmaceutical Technology Europe,September 1999.

Alternatively, the epidermal or transdermal vaccination route is alsocontemplated in the present invention.

The teaching of all references in the present application, includingpatent applications and granted patents, are herein fully incorporatedby reference. Any patent application to which this application claimspriority is incorporated by reference herein in its entirety in themanner described herein for publications and references.For the avoidance of doubt the terms ‘comprising’, ‘comprise’ and‘comprises’ herein is intended by the inventors to be optionallysubstitutable with the terms ‘consisting of’, ‘consist of’, and‘consists of’, respectively, in every instance.The invention will be further described by reference to the following,non-limiting, examples:

EXAMPLES Example 1: Design and Construction of ECD-TMD-FoldonHemagglutinin Antigen (HA) Plasmid

Hemagglutinin (HA) antigen was modified based on a native HA protein ofA/Indonesia/05/2005 H5N1 strain. At the N-terminus, the HA signalpeptide was replaced by gp67, which is a very effectivebaculovirus-encoded signal sequence for protein secretion (Whitford etal 1989, J. Virol. 63, 1393-1399). Transmembrane domain (TMD), neededfor the rosette formation, as well as trimerization domain Foldon (frombacteriophage T4 fibritin) (Meier et al 2004, J. Mol. Biol. 344,1051-1069), needed for the trimerization of HA monomers, were downstreamthe HA sequence (ECD). Finally, a poly-His tag was added at theC-terminus to facilitate the purification of the recombinant antigen.FIG. 1 shows the composition of the starting construct ECD-Foldon andthat of ECD-TMD-Foldon. ECD-Foldon was a gift from the Center forDiseases Control (CDC, Atlanta, USA). This construct was incorporated inplasmid pAcGP67, a baculovirus transfer vector containing the gp67signal sequence in front of ECD-Foldon. ECD-Foldon was used as templateto prepare the other construct and was first produced by transformationof electrocompetent TOP10 Escherichia coli cells (Life Technologies).The plasmid DNA of ECD-Foldon was purified by Maxiprep (Qiagen),according to the manufacturer's instructions. It was used to constructECD-TMD-Foldon by inserting the TM between ECD and Foldon bysite-directed mutagenesis. For that, a megaprimer was first generated byPCR. The primers consisted of sequences complementary to the pAcGP67vector together with TM sequences, and the template was HA full lengthfrom A/Indonesia/05/2005 strain in TOPO plasmid. After PCR reaction, themegaprimer was purified in agarose gel with a QIAquick gel extractionkit (Qiagen). Consecutive mutagenesis reaction was made in the followingconditions: 7.5 μl 10×reaction buffer, 2 μl dNTP (10 mM), 100 ngtemplate DNA containing ECD-Foldon; 150 ng Megaprimer, 2 μl PfuUltra HFDNA pol (2.5 U/μl), for a total volume of 50 μl. The PCR conditions were95° C. for 1 min and then 5 cycles consisting of 50 s at 95° C., 1 minat 52° C., 22 min at 68° C., followed by 13 cycles consisting of 50 s at95° C., 1 min at 55° C., and 22 min at 68° C. Thereafter, DNA templatewas removed by Dpn I digestion (Promega). PCR product was used totransform TOP10 electrocompetent cells. Miniprep purification procedurewas made on positive colonies and the validity of the construction wasconfirmed by sequencing. FIG. 2 discloses the full amino acid sequencesof ECD-Foldon and ECD-TMD-Foldon. The sequence of the recombinantfull-length HA molecule that has been used as comparator, particularlyin immunogenicity studies is also shown.

Example 2: Production and Purification of ECD-TMD-Foldon

The construct was inserted in baculovirus. For that, Hi-5 cells inSF-900 II medium containing 264 g/l NaCl were seeded in a 6-well plateat a concentration of 1×10⁶ cells/well. Fifty ng of the transfer plasmidcontaining ECD-TMD-Foldon sequence were mixed with 20 ng of BAC vector3000 and 100 μl Cellfectin reagent (Life technology; cat 10362-010) inSF-900 II medium and incubated during 30 min. Then, 800 μl of mediumwere added to the transfection mixture and the solution was distributedamong the wells. After 5 h incubation, the mixture was removed andreplaced with 2 ml of medium. Incubation occurred at 27° C. for 4 days.Cells and medium were harvested from the 6-well plates, transferred incentrifuge tubes and centrifuged to separate cells from virus-containingmedium. Supernatant was used to infect Hi-5 cells. Positive clones wereselected based on recombinant protein expression (as checked by Westernblotting) and used to generate virus stock.To purify ECD-TMD-Foldon, a total of 5-10 litres of a suspension of Hi-5cells (adjusted at 2×10⁶ cells/ml in SF500 shake flasks) was infectedwith ECD-TMD-Foldon-modified baculovirus with a multiplicity ofinfection=1. Harvest occurred 56-65 h after infection. The suspensionwas centrifuged at 200×g for 30 min and the supernatant discarded. Then200-250 ml of buffer containing 1% Triton X100 was added to each pelletfor a 30 min incubation at 4° C. on a shaking plate to resuspend thepellet. Suspension was centrifuged at 10 000×g for 25 min and thesupernatant containing ECD-TMD-Foldon was kept. RecombinantECD-TMD-Foldon was purified from the supernatant by ion exchangechromatography through an anion exchange column (Q sepharose Fast Flow,Amersham) followed by a cation exchange column (SP sepharose Fast Flow,Amersham). Recombinant molecule bound in SP column was eluted by pHchange (5.9 to 7.2) and molarity change (+150 mM NaCl). Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used toidentify the fractions of interest containing the target recombinantmolecule. Finally, a pool of the selected interesting fractions was runthrough a nickel column to further purify ECD-TMD-Foldon that displays apoly-His tag able to bind nickel. Equilibration buffer was 50 mM Tris,pH 8.0-150 mM NaCl and 10 mM imidazole. Fractions were eluted withincreasing concentrations of imidazole. Fractions of interest wereselected after run in SDS-PAGE. Imidazole was eliminated by dialysis andthe sample concentration was adjusted to 1 mg/ml.

Example 3: Characterisation (Including Rosette Formation) ofECD-TMD-Foldon

After purification, full-length HA, ECD-Foldon and ECD-TMD-Foldon werecompared by electron microscopy (EM). The samples were prepared for EMnegative staining analysis according to a standard two-step negativestaining method using phosphotungstic acid as contrasting agent. A glowdischarge was applied on the grids to improve the adsorption of thematerial on the grids.Briefly, a nickel grid (400-mesh) with carbon-coated formvar film wasfloated on a drop of the sample for 10 min at room temperature to allowadsorption of the material. Excess solution was removed. The grid wasbriefly floated on a drop of distilled water to remove salt excess andwas then transferred on a drop of stain prepared as follows: 2% (w/v) Naphosphotungstate in water supplemented with 1% trehalose (w/v). The gridwas blotted dry after 30 s. The material was left to dry completely andexamined by transmission electron microscopy under a LEO Zeiss EM91252at 100 kV.The results are shown in FIG. 3A, 3B and 3C. ECD-Foldon appeared asstructures of approximately 10 nm. In contrast, both full-length HA andECD-TMS-Foldon microphotographs showed components ranging from 10 to 50nm. This indicates that ECD-TMD-Foldon may assemble as a trimericstructure and can form rosette, similarly to full-length HA.

Example 4: ECD-TMD-Foldon is Immunogenic and Elicits NeutralisingAntibodies

The major drawback of the ECD-Foldon construct was its very poorimmunogenicity, compared with split HA antigen. One of the criticalfactor for immunogenicity of recombinant HA antigens is the possibilityto oligomerize and form rosettes. Rosette formation results from theinteraction of several HA molecules through their transmembrane domain.Therefore, the transmembrane domain has been inserted in ECD-Foldon withthe aim of allowing the formation of rosette and increaseimmunogenicity, resulting in the ECD-TMD-Foldon construct.To evaluate immunogenicity of the different constructs female C57Bl/6mice (8-10-week old; 5, 10 or 11 mice/group)) were immunized at Day 0and Day 21 with ECD-Foldon (6 or 24 μg), ECD-TMD-Foldon (1.5, 6 or 24μg), recombinant full-length HA (1.5, 6 or 24 μg), A/Indonesia/05/2005split antigen (1.5 μg) or phosphate-buffered saline (PBS) negativecontrol. All antigens were adjuvanted with AS03, an Adjuvant Systemcontaining α-tocopherol and squalene in an oil-in-water emulsion (Garçonet al 2012, Expert. Rev. Vaccines 11, 349-366).Blood was taken on Days 21 (3 weeks after dose I) and 42 (3 weeks afterdose II). Anti-A/Indonesia/05/2005 antibodies were determined byenzyme-linked immunosorbent assay (ELISA). For that, microtiter plateswere coated overnight with A/Indonesia/05/2005 split antigen (1 μg/ml inDulbecco's phosphate-buffered saline [DPBS]) at 4° C. After saturationof the aspecific sites by a saturation buffer (without serum), serialtwo-fold dilutions of the mouse sera were added to the wells andincubated for 1.5 h at 37° C. Dilutions were done in the saturationbuffer. After wells were washed with PBS-Tween 0.1%,peroxidase-conjugated anti-mouse IgG antibody (Sigma, A5278) was addedand incubated for 1 h at 37° C. Bound antibodies were revealed by theaddition of the peroxidase substrate orthophenylenediamine in thepresence of hydrogen peroxide for 20 min at room temperature in thedark. The colorimetric reaction was stopped by addition of sulphuricacid 2 N and plates were read in a microtiter plate reader. Results areshown in FIG. 4. It was observed that ECD-TMD-Foldon is more immunogenicthan ECD-Foldon, but it does not elicit as high levels of antibodies asthe corresponding split antigen.The humoral immune responses were also evaluated by measurement ofhaemagglutination-inhibition antibody (HI) titers. The day before theassay, 50 μl of receptor destroying enzyme (RDE; cholera filtrate; SigmaC-8772), diluted at 25% in DPBS, was added to 12.5 μl of mouse serumsample and the mixture was incubated 18 h at 37° C. Then, 37.5 μl ofsodium citrate 2.5% was added to the mixture and incubated 30 min at 56°C., and DPBS was added for a total volume of 125 μl. Aftercentrifugation, supernatant was diluted 1/10th and serial twofolddilutions of it were pipetted in a microtiterplate. To each well wasadded 25 μl of a virus suspension (4 UHA in DPBS) for an incubation of30 min at room temperature. Finally, 50 μl of equine erythrocytes (1% inDPBS) was added and incubated for 1.5-2 h at room temperature. Resultsare shown in FIG. 5. All ECD-TMD-Foldon, recombinant full-length HA andsplit antigen groups were different from the two ECD-Foldon groups andnot different from each other 3 weeks after the second immunization(One-Way ANOVA followed by Tuckey-HSD post-test).The functionality of the immunization-induced antibodies was assessed byin vitro flu neutralization assay. For that, mouse serum sample wasserially diluted twofold and dilutions were dropped in a microtiterplate (100 μl/well). To the samples, 50 μl of viral solution (2000TCID₅₀/ml) was added and the mixture was incubated for 1.5 h at roomtemperature. After incubation, 100 μl of Madin-Darby canine kidney(MDCK) cell suspension (2.4×10⁵ cells /ml cell culture medium) was addedper well. The microtiter plate was placed in an incubator (35° C. with5% CO₂) for 5-7 days. After incubation, 50 μl of the supernatants ineach well were transferred in another microtiter plate. To each well, 50μl of chicken erythrocytes (0.5% in PBS) were added and incubated for 1h at room temperature. Results are shown in FIG. 6. All ECD-TMD-Foldon,recombinant full-length HA and split antigen groups were different fromthe two ECD-Foldon groups and not different from each other 3 weeksafter the second immunization (One-Way ANOVA followed by Tuckey-HSDpost-test).FIGS. 7 & 8 show the HI and neutralizing activities, respectively,relatively to the levels of antibodies. The ratios for ECD-TMS-Foldonare in the same range as the ratios obtained for the split antigen.

Sequence ListingSEQ ID NO: 1: Amino acid sequence of recombinant H5 hemagglutinin (ECD-TMD-foldon) Indo05DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGBSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSPMFHNIHPLTIGECPKYBKSNRLVLATGLRNSPQRESRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMMAGLSLWMCSSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHSEQ ID NO: 2 nucleic acid sequence of recombinant H5 hemagglutinin (ECD-TMD-foldon)GATCCCGGGGATCAGATTTGCATTGGTTACCATGCAAACAATTCAACAGAGCAGGTTGACACAATCATGGAAAAGAACGTTACTGTTACACATGCCCAAGACATACTGGAAAAGACACACAACGGGAAGCTCTGCGATCTAGATGGAGTGAAGCCTCTAATTTTAAGAGATTGTAGTGTAGCTGGATGGCTCCTCGGGAACCCAATGTGTGACGAATTCATCAATGTACCGGAATGGTCTTACATAGTGGAGAAGGCCAATCCAACCAATGACCTCTGTTACCCAGGGAGTTTCAACGACTATGAAGAACTGAAACACCTATTGAGCAGAATAAACCATTTTGAGAAAATTCAAATCATCCCCAAAAGTTCTTGGTCCGATCATGAAGCCTCATCAGGAGTGAGCTCAGCATGTCCATACCTGGGAAGTCCCTCCTTTTTTAGAAATGTGGTATGGCTTATCAAAAAGAACAGTACATACCCAACAATAAAGAAAAGCTACAATAATACCAACCAAGAAGATCTTTTGGTACTGTGGGGAATTCACCATCCTAATGATGCGGCAGAGCAGACAAGGCTATATCAAAACCCAACCACCTATATTTCCATTGGGACATCAACACTAAACCAGAGATTGGTACCAAAAATAGCTACTAGATCCAAAGTAAACGGGCAAAGTGGAAGGATGGAGTTCTTCTGGACAATTTTAAAACCTAATGATGCAATCAACTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGCATACAAAATTGTCAAGAAAGGGGACTCAGCAATTATGAAAAGTGAATTGGAATATGGTAACTGCAACACCAAGTGTCAAACTCCAATGGGGGCGATAAACTCTAGTATGCCATTCCACAACATACACCCTCTCACCATCGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCTTGCAACAGGGCTCAGAAATAGCCCTCAAAGAGAGAGCAGAAGAAAAAAGAGAGGACTATTTGGAGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATGAGCAGGGGAGTGGGTACGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAGTCACCAATAAGGTCAACTCAATCATTGACAAAATGAACACTCAGTTTGAGGCCGTTGGAAGGGAATTTAATAACTTAGAAAGGAGAATAGAGAATTTAAACAAGAAGATGGAAGACGGGTTTCTAGATGTCTGGACTTATAATGCCGAACTTCTGGTTCTCATGGAAAATGAGAGAACTCTAGACTTTCATGACTCAAATGTTAAGAACCTCTACGACAAGGTCCGACTACAGCTTAGGGATAATGCAAAGGAGCTGGGTAACGGTTGTTTCGAGTTCTATCACAAATGTGATAATGAATGTATGGAAAGTATAAGAAACGGAACGTACAACTATCCGCAGTATTCAGAAGAAGCAAGACTAAAAAGAGAGGAAATAAGTGGGGTAAAATTGGAATCAATAGGAACTTACCAAATACTGTCAATTTATTCAACAGTGGCGAGTTCCCTAGCACTGGCAATCATGATGGCTGGTCTATCTTTATGGATGTGCTCCAGCGGCCGCTTGGTCCCTCGTGGAAGCCCAGGCTCCGGCTACATCCCCGAGGCCCCGCGCGACGGCCAGGCCTACGTGCGCAAGGACGGCGAGTGGGTGCTGCTGTCCACCTTCCTGGGACATCATCATCATCATCATTGASEQ ID NO: 3 amino acid sequence of the intracellular domain of H5 hemagglutininNGSLQCRICISEQ ID NO: 4 nucleic acid sequence of the intracellular domain of H5 hemagglutininAATGGATCGTTACAATGCAGAATTTGCATTSEQ ID NO: 5 amino acid sequence of the transmembrane domain of H5 HAGVKLESIGTYQILSIYSTVASSLALAIMMAGLSLWMCSSEQ ID NO: 6 nucleic acid sequence of the transmembrane domain of H5 HAGGGGTAAAATTGGAATCAATAGGAACTTACCAAATACTGTCAATTTATTCAACAGTGGCGAGTTCCCTAGCACTGGCAATCATGATGGCTGGTCTATCTTTATGGATGTGCTCCSEQ ID NO: 7 amino acid sequence of extracellular domain of H5 HADQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGBSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKREEISSEQ ID NO: 8 nucleic acid sequence of extracellular domain of H5 HAGATCCCGGGGATCAGATTTGCATTGGTTACCATGCAAACAATTCAACAGAGCAGGTTGACACAATCATGGAAAAGAACGTTACTGTTACACATGCCCAAGACATACTGGAAAAGACACACAACGGGAAGCTCTGCGATCTAGATGGAGTGAAGCCTCTAATTTTAAGAGATTGTAGTGTAGCTGGATGGCTCCTCGGGAACCCAATGTGTGACGAATTCATCAATGTACCGGAATGGTCTTACATAGTGGAGAAGGCCAATCCAACCAATGACCTCTGTTACCCAGGGAGTTTCAACGACTATGAAGAACTGAAACACCTATTGAGCAGAATAAACCATTTTGAGAAAATTCAAATCATCCCCAAAAGTTCTTGGTCCGATCATGAAGCCTCATCAGGAGTGAGCTCAGCATGTCCATACCTGGGAAGTCCCTCCTTTTTTAGAAATGTGGTATGGCTTATCAAAAAGAACAGTACATACCCAACAATAAAGAAAAGCTACAATAATACCAACCAAGAAGATCTTTTGGTACTGTGGGGAATTCACCATCCTAATGATGCGGCAGAGCAGACAAGGCTATATCAAAACCCAACCACCTATATTTCCATTGGGACATCAACACTAAACCAGAGATTGGTACCAAAAATAGCTACTAGATCCAAAGTAAACGGGCAAAGTGGAAGGATGGAGTTCTTCTGGACAATTTTAAAACCTAATGATGCAATCAACTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGCATACAAAATTGTCAAGAAAGGGGACTCAGCAATTATGAAAAGTGAATTGGAATATGGTAACTGCAACACCAAGTGTCAAACTCCAATGGGGGCGATAAACTCTAGTATGCCATTCCACAACATACACCCTCTCACCATCGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCTTGCAACAGGGCTCAGAAATAGCCCTCAAAGAGAGAGCAGAAGAAAAAAGAGAGGACTATTTGGAGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATGAGCAGGGGAGTGGGTACGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAGTCACCAATAAGGTCAACTCAATCATTGACAAAATGAACACTCAGTTTGAGGCCGTTGGAAGGGAATTTAATAACTTAGAAAGGAGAATAGAGAATTTAAACAAGAAGATGGAAGACGGGTTTCTAGATGTCTGGACTTATAATGCCGAACTTCTGGTTCTCATGGAAAATGAGAGAACTCTAGACTTTCATGACTCAAATGTTAAGAACCTCTACGACAAGGTCCGACTACAGCTTAGGGATAATGCAAAGGAGCTGGGTAACGGTTGTTTCGAGTTCTATCACAAATGTGATAATGAATGTATGGAAAGTATAAGAAACGGAACGTACAACTATCCGCAGTATTCAGAAGAAGCAAGACTAAAAAGAGAGGAAATAAGTSEQ ID NO: 9 amino acid sequence of T4 bacteriophage fibritin “foldon”GSGYIPEAPRDGQAYVRKDGEWVLLSTFLSEQ ID NO: 10 nucleic acid sequence of T4 bacteriophage fibritin “foldon”GGCTCCGGCTACATCCCCGAGGCCCCGCGCGACGGCCAGGCCTACGTGCGCAAGGACGGCGAGTGGGTGCTGCTGTCCACCTTCCTG SEQ ID NO: 11 amino acid sequence of full length HADQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGBSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMMAGLSTWMCSNGSLQCRICILVPRGSHHHHHH

1-27. (canceled)
 28. A method for producing a recombinant influenzavirus hemagglutinin (HA) antigen comprising the extracellular domain ofHA or an immunogenic portion thereof, a hydrophobic signal and aheterologous trimerisation domain comprising expressing a polynucleotideencoding said recombinant influenza virus hemagglutinin (HA) antigen ina eukaryotic cell, such as a mammalian cell, e.g. a CHO cell, or aninsect cell, optionally further comprising purifying/isolating therecombinant HA from the eukaryotic cell.
 29. A method for preventionand/or vaccination against influenza disease, comprising theadministration a recombinant influenza virus hemagglutinin (HA) antigencomprising the extracellular domain of HA or an immunogenic portionthereof, followed by a hydrophobic signal, followed by a heterologoustrimerisation domain to a person in need thereof.
 30. The method ofclaim 29, wherein less than 15 micrograms of hemagglutinin areadministered per dose.
 31. The method of claim 30, wherein 3.75 to 10micrograms of hemagglutinin are administered per dose.
 32. The method ofclaim 29, wherein the hydrophobic signal comprises a stretch of at least5 hydrophobic amino acids.
 33. The method of claim 29, wherein thehydrophobic signal is an HA transmembrane domain.
 34. The method ofclaim 29, wherein the HA antigen lacks the intracellular domain ofinfluenza hemagglutinin.
 35. The method of claim 29, wherein thehemagglutinin is not full length hemagglutinin.
 36. The method of claim29, wherein the recombinant HA antigen forms rosette structures in vivoor in vitro.
 37. The method of claim 29, which additionally comprises acleavable linkage.
 38. The method of claim 29, wherein the hemagglutininis from an H1, H2, H3, H5, H7 or H9 strain.
 39. The method of claim 29,wherein the HA transmembrane is heterologous to the extracellular domainof said HA.
 40. The method of claim 29, wherein i) the HA ECD consistsof or comprises SEQ ID NO: 7, ii) the HA TMD consists of or comprisesSEQ ID NO: 5 or a fragment or derivative of this sequence that retainsthe ability to orientate HA trimers into rosette structures andmaintains the immunogenicity of HA and/or iii) the trimerisation domainconsists of or comprises SEQ ID NO: 9 or a derivative of this sequencethat maintains the ability to induce rHA monomers to form trimers. 41.The method of claim 29, wherein i) the HA ECD consists of or comprisesSEQ ID NO: 7, ii) the HA TMD consists of or comprises SEQ ID NO:5 andiii) the trimerisation domain consists of or comprises SEQ ID NO:
 9. 42.The method of claim 29, wherein the HA antigen comprises the sequence ofSEQ ID NO:1.
 43. The method of claim 29, wherein the hemagglutinin lacksthe HA stalk, or part of the stalk.
 44. The method of claim 29,comprising administration of an immunogenic composition comprising thehemagglutinin (HA) antigen and a pharmaceutically-acceptable carrier.45. The method of claim 44, wherein the immunogenic composition furthercomprises an adjuvant.
 46. The method of claim 45, wherein the adjuvantis an oil-in-water emulsion adjuvant.
 47. The method of claim 44,wherein the composition is multivalent or monovalent.